Method of treating and/or preventing inflammation by administering olanexidine

ABSTRACT

An object of the present invention is to provide a composition that can be used as a novel anti-inflammatory agent. An inflammation such as stomatitis, oral mucositis, gingivitis, or pneumonia can be ameliorated and/or prevented by using a composition comprising olanexidine or a pharmacologically acceptable salt thereof. The composition of the present invention preferably further comprises a poloxamer which is a block copolymer consisting of a chain of polyoxypropylene (POP) and two chains of polyoxyethylene (POE) flanking the POP.

TECHNICAL FIELD

The present invention relates to an anti-inflammatory agent comprisingolanexidine or a pharmacologically acceptable salt thereof as an activeingredient.

BACKGROUND ART

Olanexidine is a compound, called1-(3,4-dichlorobenzyl)-5-octylbiguanide under chemical name, having highbactericidal activity. Olanexidine gluconate, which is a gluconatethereof, has a wide bactericidal spectrum. Its bactericidal effectappears in a short time, and further, the activity persists for a longtime. Moreover, an aqueous solution of olanexidine gluconate is highlystable, can be preserved for a long period, furthermore is low irritantor toxic to the skin, and is also excellent in safety. In addition, theaqueous solution of olanexidine gluconate is free from problems withcolor, odor and taste and as such, is easily produced as drugformulation (patent document 1). Hence, olanexidine gluconate is mainlyused in the antisepsis of the skin at an operation site (field ofoperation).

However, it has not been known so far that olanexidine or a salt thereofexhibits anti-inflammatory action. Moreover, since olanexidine gluconatehas irritancy to the mucosa, the olanexidine gluconate is difficult toapply to the mucosa such as the oral mucosa.

PRIOR ART DOCUMENT

Patent Document

-   Patent document 1: Japanese unexamined Patent Application    Publication No. 2005-289959

SUMMARY OF THE INVENTION Object to be Solved by the Invention

An object of the present invention is to provide a composition that canbe used as a novel anti-inflammatory agent.

Means to Solve the Object

The present inventors have conducted diligent studies to attain theobject and consequently found that, unexpectedly, olanexidine or a saltthereof exhibits anti-inflammatory action. The present inventors havefurther found that olanexidine gluconate is applicable to the mucosasuch as the oral mucosa by using a composition comprising theolanexidine gluconate and a poloxamer which is a block copolymerconsisting of a chain of polyoxypropylene (POP) and two chains ofpolyoxyethylene (POE) flanking the POP, leading to the completion of thepresent invention.

Specifically, the present invention is as follows.

(1) A composition for amelioration and/or prevention of an inflammation,comprising olanexidine or a pharmacologically acceptable salt thereof.

(2) The composition according to (1), wherein the olanexidine or thepharmacologically acceptable salt thereof is olanexidine gluconate.

(3) The composition according to (1) or (2), further comprising apoloxamer which is a block copolymer consisting of a chain ofpolyoxypropylene (POP) and two chains of polyoxyethylene (POE) flankingthe POP.

(4) The composition according to any one of (1) to (3), wherein theinflammation is selected from stomatitis, oral mucositis, gingivitis,and pneumonia.

(5) The composition according to any one of (1) to (4), wherein

the inflammation is oral mucositis due to treatment of a cancer, and

the composition comprises

0.01 to 1.5% (W/V) of olanexidine gluconate, and

a poloxamer which is a block copolymer consisting of a chain ofpolyoxypropylene (POP) and two chains of polyoxyethylene (POE) flankingthe POP.

(6) The composition according to any one of (3) to (5), wherein thepoloxamer is selected from polyoxyethylene (42) polyoxypropylene (67)glycol (Pluronic P-123), polyoxyethylene (54) polyoxypropylene (39)glycol (Pluronic P-85), and polyoxyethylene (196) polyoxypropylene (67)glycol (Pluronic F-127).(7) The composition according to (6), wherein the poloxamer ispolyoxyethylene (42) polyoxypropylene (67) glycol (Pluronic P-123).(8) The composition according to any one of (1) to (7), wherein aconcentration of the olanexidine gluconate is 0.05 to 0.5% (W/V).(9) The composition according to any one of (3) to (8), wherein aconcentration of the poloxamer is 0.1 to 5.0% (W/V).(10) The composition according to any one of (1) to (9), wherein thecomposition is in a form of a liquid or a gargle.(11) The composition according to any one of (5) to (10), wherein thetreatment of the cancer is chemotherapy, radiotherapy, or concurrentchemoradiotherapy.

Other examples of the mode of carrying out the present invention caninclude a method for ameliorating or preventing (treating) aninflammation by administering the composition for amelioration and/orprevention of an inflammation of the present invention to a patient inneed of amelioration or prevention (treatment) of an inflammation, amethod for ameliorating or preventing (treating) oral mucositis due totreatment of a cancer by administering the composition for ameliorationand/or prevention of oral mucositis due to treatment of a cancer of thepresent invention to a patient in need of amelioration or prevention(treatment) of oral mucositis due to treatment of a cancer, acomposition comprising olanexidine or a pharmacologically acceptablesalt thereof for use in amelioration or prevention (treatment) of aninflammation, a composition comprising 0.01 to 1.5% (W/V) of olanexidinegluconate, and a poloxamer which is a block copolymer consisting of achain of polyoxypropylene (POP) and two chains of polyoxyethylene (POE)flanking the POP for use in amelioration or prevention (treatment) oforal mucositis due to treatment of a cancer, use of olanexidine or apharmacologically acceptable salt thereof for preparing the compositionfor amelioration and/or prevention of an inflammation of the presentinvention, and use of 0.01 to 1.5% (W/V) of olanexidine gluconate, and apoloxamer which is a block copolymer consisting of a chain ofpolyoxypropylene (POP) and two chains of polyoxyethylene (POE) flankingthe POP for preparing the composition for amelioration and/or preventionof oral mucositis due to treatment of a cancer of the present invention.

Effect of the Invention

The present invention provides a novel composition for ameliorationand/or prevention of an inflammation. The composition of the presentinvention is applicable to a wide range of inflammations such asstomatitis, oral mucositis, gingivitis, and pneumonia. Moreover, thecomposition for amelioration and/or prevention of an inflammation(anti-inflammatory agent) of the present invention can ameliorate and/orprevent oral mucositis in a patient who is receiving chemotherapy,radiotherapy, or concomitant chemotherapy and radiotherapy of a cancer,and thus, can prevent reduction in QOL, such as inhibition of acommunication function, sleep disorder, pain, or dysphagia (decreaseddietary intakes), in a patient, or disturbance of dose conformity ofchemotherapy and/or radiotherapy.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing results of measuring the number of bacteriain the oral cavity by aerobic culture in Example 1. The number ofbacteria in the ordinate was indicated by a logarithmic value.

FIG. 2 is a diagram showing results of measuring the number of bacteriain the oral cavity by culture in a streptococcus selective medium inExample 1. The number of bacteria in the ordinate was indicated by alogarithmic value.

FIG. 3 is a diagram showing results of measuring the number of bacteriain the oral cavity by anaerobic culture in Example 1. The number ofbacteria in the ordinate was indicated by a logarithmic value.

FIG. 4 is a diagram showing results of measuring the number of bacteriain the oral cavity using a bacterial counter in Example 1. The number ofbacteria in the ordinate was indicated by a logarithmic value.

FIG. 5 is a diagram showing results of a comparison test betweenOlanedine® antiseptic solutions (OPB) and other agents in Example 2. Thenumber of bacteria in the ordinate was indicated by a logarithmic value.

FIG. 6 is a diagram showing results of studying the influence of anolanexidine concentration on bactericidal efficacy in Example 3. Thenumber of bacteria in the ordinate was indicated by a logarithmic value.

FIG. 7 is a diagram showing change in body weight of each group inExample 4.

FIG. 8 is a table showing macroscopic observation results in Example 4.The numeric values in the table represent stomatitis grades, and theshaded areas represent groups presenting with a leukoplakia-like symptom(increased keratosis, thickening, etc.).

FIG. 9 is a photograph of the cheek pouch of each group on the final dayin Example 4.

FIG. 10 is a table showing histopathological examination results inExample 4.

FIG. 11 is a table showing macroscopic observation results in Example 5.The numeric values in the table represent stomatitis grades, and theshaded areas represent groups presenting with a leukoplakia-like symptom(increased keratosis, thickening, etc.).

FIG. 12 is a table showing histopathological examination results inExample 5.

FIG. 13 is a diagram showing the number of surviving bacteria in thehamster oral cavity in Example 6. The number of bacteria in the ordinatewas indicated by a logarithmic value.

FIG. 14 is a diagram showing a stomatitis grade in Example 6. Thestomatitis grade is shown in the ordinate.

FIG. 15 is a diagram showing a stomatitis grade in Example 7. Thestomatitis grade is shown in the ordinate.

FIG. 16 is a diagram showing a stomatitis grade in Example 8. Thestomatitis grade is shown in the ordinate.

FIG. 17 is a diagram showing pathological examination results in Example9.

FIG. 18 is a micrograph of a HE-stained specimen in Example 9.

FIG. 19 is a diagram showing results of hematological examination andbiochemical examination in Example 10.

FIG. 20 is a diagram showing the inhibition rate of SEAP expression byan Olanedine® antiseptic solution (OPB) in Example 11.

FIG. 21 is a diagram showing the inhibition of NO production by anOlanedine® antiseptic solution (OPB) in Example 12.

MODE OF CARRYING OUT THE INVENTION

The composition of the present invention is a composition foramelioration and/or prevention of an inflammation, comprisingolanexidine or a pharmacologically acceptable salt thereof. A saltpharmacologically known in the art can be used as the pharmacologicallyacceptable salt of olanexidine. Examples thereof can includehydrochloride, carbonate, bicarbonate, citrate, gluconate, lactate,acetate, gluceptate, and tartrate. Olanexidine gluconate is preferredfrom the viewpoint of solubility in water.

In the composition of the present invention, olanexidine can becontained at a concentration that can exhibit anti-inflammatory action.Examples thereof can include 0.001 to 20% (W/V), preferably 0.005 to 15%(W/V), more preferably 0.01 to 10% (W/V), further preferably 0.1 to 5%(W/V), in terms of olanexidine gluconate. In the case of applying thecomposition of the present invention to the mucosa such as the oralmucosa, the concentration of olanexidine is preferably 0.01 to 1.5%(W/V), more preferably 0.05 to 0.5% (W/V), further preferably 0.1 to0.3% (W/V), in terms of olanexidine gluconate. In the case of applyingthe composition of the present invention to the oral mucosa, it is notdesirable that bactericidal efficacy on oral bacteria cannot besufficiently obtained if the concentration of olanexidine gluconate islower than 0.01% (W/V), and irritation to the oral mucosa is too strongif the concentration of olanexidine gluconate exceeds 1.5% (W/V).

The composition of the present invention may further comprise one ormore poloxamers in order to reduce irritation to an application site. Inthis context, the poloxamer is not particularly limited as long as thepoloxamer is a block copolymer consisting of a chain of polyoxypropylene(POP) and two chains of polyoxyethylene (POE) flanking the POP, andreduces irritation to an application site. One or more poloxamersselected from polyoxyethylene (42) polyoxypropylene (67) glycol(Pluronic P-123), polyoxyethylene (54) polyoxypropylene (39) glycol(Pluronic P-85), and polyoxyethylene (196) polyoxypropylene (67) glycol(Pluronic F-127) are preferred. Among others, examples thereof caninclude polyoxyethylene (42) polyoxypropylene (67) glycol (PluronicP-123), polyoxyethylene (3) polyoxypropylene (17) glycol (PluronicL-31), polyoxyethylene (20) polyoxypropylene (20) glycol (PluronicL-44), polyoxyethylene (120) polyoxypropylene (40) glycol (PluronicF-87), and polyoxyethylene (160) polyoxypropylene (30) glycol (PluronicF-68).

Among the poloxamers described above, one or more poloxamers selectedfrom polyoxyethylene (42) polyoxypropylene (67) glycol (Pluronic P-123),polyoxyethylene (54) polyoxypropylene (39) glycol (Pluronic P-85), andpolyoxyethylene (196) polyoxypropylene (67) glycol (Pluronic F-127) arepreferred. Among them, polyoxyethylene (42) polyoxypropylene (67) glycol(Pluronic P-123) is more preferred.

Examples of the concentration of the poloxamer can include, but are notparticularly limited to, 0.1 to 5.0% (W/V), preferably 0.1 to 4.0%(W/V), more preferably 0.1 to 3.0% (W/V), further preferably 0.1 to 2.0%(W/V), most preferably 0.1 to 1.5% (W/V). The concentration ratiobetween olanexidine gluconate and the poloxamer is preferably 1:2 to1:20, more preferably 1:5 to 1:10. In the case of applying thecomposition of the present invention to the oral mucosa, irritation ofthe oral mucosa by olanexidine gluconate is strong in a highconcentration range equal to or higher than an olanexidine gluconateconcentration of 0.3% (W/V). Therefore, a larger amount of the poloxameris more preferred for suppressing irritation (increased keratosis) byolanexidine gluconate.

In the present specification, the “inflammation” means biologicalreaction causing a sign such as flare, a feeling of warmth, swelling, orpain due to an internal factor such as autoimmune disease, or anexternal factor such as bacterial or viral infection, trauma, physicalirritation (heat, coldness, radiation, electricity, etc.), or a chemicalsubstance. The inflammation according to the present invention is notparticularly limited as long as the composition of the present inventioncan be applied to the inflammation. Examples thereof can preferablyinclude an inflammation involving a Toll-like receptor, more preferablyan inflammation due to bacterial infection. Examples of the inflammationsite can include the brain, the eye, the trachea, a vascular vessel, thelung, the liver, the heart, the pancreas, the stomach, the intestine,the mesenterium, the kidney, the skin, the nasal mucosa, the oralmucosa, the gingiva and the joint. Specific examples of the inflammationcan include encephalitis, bronchitis, angiitis, pneumonia, hepatitis,myocarditis, pancreatitis, enteritis, gastritis, peritonitis, nephritis,stomatitis, oral mucositis, gingivitis, arthritis, an inflammationcaused by reperfusion injury after ischemia, an inflammation caused byimmune rejection after transplantation, an inflammation caused by burnor multiple organ failure, inflammation developed after operation, andan inflammation caused by arteriosclerosis. Among them, preferredexamples thereof can include stomatitis, oral mucositis, gingivitis, andpneumonia. In the present specification, the oral mucositis refers to aninflammation developed in the oral mucosa by treatment of a cancer, andthe stomatitis refers to an inflammation developed in the oral mucosaindependently of treatment of a cancer. Alternatively, the compositionof the present invention may be a composition having a specific purposeof ameliorating and/or preventing oral mucositis due to treatment of acancer. In one aspect, the present invention excludes a compositionhaving a purpose of ameliorating and/or preventing oral mucositis due totreatment of a cancer.

The composition of the present invention can be applied to the skin, theoral mucosa, or the mucosa of the gingiva, the gastrointestinal tract,the trachea, the lung, or the like at an inflammation site. Examples ofthe administration method can include injection (intravenous,intramuscular, subcutaneous, intracutaneous, intraperitoneal, etc.),oral administration, percutaneous administration, inhalation,embrocation to the oral cavity, embrocation to the gingiva, andgargling. Preparations can be appropriately produced according to theseadministration methods. A selectable dosage form is not particularlylimited, and the dosage form can be widely selected from, for example,an injection (a solution, a suspension, an emulsion, a solid formulationfor dissolution in use, etc.), a tablet, a capsule, a granule, a powder,a liquid, a gargle, a liposome formulation, an ointment, a gel, a powerfor external use, a spray, and an inhalation powder. Also, componentsusually used in medicaments, such as a common excipient, stabilizer,binder, lubricant, emulsifier, osmotic pressure adjuster, pH adjuster,colorant, and disintegrant can be used for preparing these drugformulations.

In the case of applying the composition of the present invention to theoral cavity, any dosage form suitable for application to the oral cavitymay be used. Preferred examples thereof can include a liquid and agargle. Alternatively, a solid composition gradually dissolving ordisintegrating in the mouth, such as lozenges, candies, gummy candies,troches, or gums, may be used. Moreover, if necessary, the compositionof the present invention can further contain various additives that areused for the purpose of conferring flavor or coloring. Examples of theadditive for the purpose of conferring flavor can include a syntheticfragrance, a natural fragrance, and a sweetener such as aspartame,acesulfame potassium, sucralose, alitame, neotame, a licorice rootextract (glycyrrhizin), saccharin, saccharin sodium, a stevia extract,and a stevia powder. Examples of the additive for the purpose ofcoloring can include caramel, a natural coloring agent, and a syntheticcoloring agent. Also, the composition of the present invention maycontain an additive such as an emulsifier (glycerin fatty acid ester,sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrosefatty acid ester, lecithin, etc.), a stabilizer, or a preservative.These additive agents may be used alone or in combination of two or morethereof.

In the case of administering the composition of the present invention asa liquid or a gargle, a single dose can be arbitrarily determineddepending on a site where an inflammation has been developed, orseverity. Examples thereof can include 1 to 100 mL, preferably 2 to 50mL, more preferably 5 to 40 mL, most preferably 10 to 30 mL.

The timing of administration of the composition of the present inventioncan be arbitrarily determined depending on a site where an inflammationhas been developed, severity, or the degree of amelioration of aninflammation. Examples thereof can include after eating, after wake-up,and before bedtime. Alternatively, the composition of the presentinvention may be administered at intervals of 2 to 8 hours, preferablyat intervals of 4 to 6 hours. Also, the composition of the presentinvention can prevent an inflammation by administration to a patientbefore operation or a patient after oral care.

The administration period of the composition of the present inventioncan be arbitrarily determined depending on the degree of amelioration ofan inflammation. Examples thereof can include 1 week to 3 months,preferably 1 week to 2 months, more preferably 1 week to 1 month, mostpreferably 1 to 2 weeks.

In the present invention, examples of the treatment of the cancer caninclude chemotherapy, radiotherapy, and concurrent chemoradiotherapy ofthe cancer. The chemotherapy of the cancer refers to general treatmentof the cancer with an anticancer agent. Examples of the anticancer agentused in the present invention can include a pyrimidine fluoride-basedantimetabolite such as fluorouracil (5-FU), tegafur/gimeracil/oteracilpotassium (S-1), and tegafur/uracil (UFT), a folate antagonist such asmethotrexate, an antitumor antibiotic such as daunorubicin, doxorubicin,epirubicin, bleomycin, peplomycin, and actinomycin D, a vegetablealkaloid such as paclitaxel, docetaxel, vincristine, and etoposide, anda platinum-containing drug such as cisplatin, carboplatin, andnedaplatin, which easily cause oral mucositis. Particularly preferredexamples thereof can include a pyrimidine fluoride-based antimetabolitesuch as 5-FU. These anticancer agents may be used alone or incombination of two or more thereof.

In the present invention, the radiotherapy is a treatment for thepurpose of suppressing proliferation of cancer cells by irradiating amalignant tumor portion with radiation. Examples of the radiation foruse in the treatment include an X-ray and an electron beam. Theconcurrent chemoradiotherapy refers to a treatment method that enhancesthe effect of radiation by using radiation therapy, which is a localcancer therapy, and an anticancer agent in combination. The target siteof the radiotherapy is not particularly limited. Examples of theradiotherapy can include radiotherapy in the head and neck portion,particularly, in the oral cavity or the pharyngeal portion.

Hereinafter, the present invention will be described more specificallywith reference to Examples. However, the technical scope of the presentinvention is not limited by these examples.

Example 1

1. Test on Bactericidal Efficacy in Oral Cavity Using Cynomolgus Monkey

In this test, bactericidal efficacy on bacteria in the oral cavity ofcynomolgus monkeys was compared and studied by using a simplifiedbacterial counter and a culture technique in combination, and using testmaterials (0.1% (w/v) olanexidine gluconate and 0.47% povidone-iodine asbactericidal antiseptics, and saline as a negative control drug).

1-1 Test Material

A test substance was prepared by diluting Olanedine® Antiseptic Solution1.5% (hereinafter, referred to as “1.5% OPB”, etc.; a solutioncontaining 1.508% (w/v) of olanexidine gluconate, manufactured by OtsukaPharmaceutical Factory, Inc.) 15-fold such that the olanexidinegluconate concentration was 0.1% (w/v) (0.1% OPB). A control substanceIsodine Gargle Solution 7% (hereinafter, referred to as “7% PVP-I”,etc.; manufactured by Meiji Seika Pharma Co., Ltd.) was diluted 15-fold(0.47% PVP-I), and saline (manufactured by Otsuka PharmaceuticalFactory, Inc.) was used as it was.

1-2 Test Animal

Cynomolgus monkeys (male, produced in Cambodia, manufactured byEveBioscience Co., Ltd.) which were 2 years and 11 months to 3 years and11 months old when used, were used.

1-2-1 Group Configuration

The oral cavity of each animal was used as a test site. Nine animalswere used, and the number of test sites per group was set to 3 in orderto embrocate 0.1% OPB, 0.47% PVP-I and saline. Bacteria were collected atotal of 4 times (before test material embrocation, and 10 minutes, 6hours and 24 hours after embrocation).

1-2-2 Animal Number and Sample Number

Animal numbers and sample numbers were assigned as shown in Table 1below. The numbers of baseline bacteria of animal Nos. 1 to 3 weremeasured, and the animals were subjected to tests using saline, 0.1%OPB, and 0.47% PVP-I in descending order of the number of bacteria.Likewise, animal Nos. 4 to 6 were subjected to tests using 0.1% OPB,0.47% PVP-I, and saline in descending order of the number of bacteria,and animal Nos. 7 to 9 were subjected to tests using 0.47% PVP-I,saline, and 0.1% OPB in descending order of the number of bacteria.

TABLE 1 Sample No. Animal No. Time point 1 1 baseline 2 10 min 3 6 hr 424 hr 5 2 baseline 6 10 min 7 6 hr 8 24 hr 9 3 baseline 10 10 min 11 6hr 12 24 hr 13 4 baseline 14 10 min 15 6 hr 16 24 hr 17 5 baseline 18 10min 19 6 hr 20 24 hr 21 6 baseline 22 10 min 23 6 hr 24 24 hr 25 7baseline 26 10 min 27 6 hr 28 24 hr 29 8 baseline 30 10 min 31 6 hr 3224 hr 33 9 baseline 34 10 min 35 6 hr 36 24 hr1-3 Testing Method1-3-1 Anesthesia

The cynomolgus monkeys were systemically anesthetized by intramuscularlyinjecting a 2:1 mixed solution of Ketalar (50 mg/mL in terms ofketamine, manufactured by Daiichi Sankyo Propharma Co., Ltd.) andSeractal 2% Injection Solution (2.0 g/100 mL in terms of xylazine,manufactured by Bayer Yakuhin, Ltd.) at 0.5 mL per kg of body weight.

1-3-2 Embrocation

[1] 100 mL of each test material was poured to a container (250 mL,manufactured by Corning Inc.) in which two Mouth Pure Oral Care Sponges(manufactured by Kawamoto Corp.) were placed.

[2] The air was evacuated from the sponges, and the sponges weresufficiently soaked in the test material.

[3] The resultant was embrocated to the oral cavity for approximately 2minutes.

1-3-3 Bacterial Collection 1

[1] Bacteria were collected from the monkey oral cavity using asterilized glove and a sterile swab (both the lateral walls in the oralcavity were scrubbed back and forth twice).

[2] The swab was placed in 5 mL of a sampling solution (10% (w/v)polysorbate 80, 0.04% (w/v) potassium dihydrogen phosphate, 0.1% (w/v)Triton X-100, 1.01% (w/v) anhydrous sodium monohydrogen phosphate, 2%(w/v) soybean lecithin, 5% (w/v) polyoxyethylene (20) cetyl ether, pH7.8 to 7.9).1-3-4 Bacterial Collection 2[1] A swab of expendable supplies for measurement (DU-AC02NP-H,manufactured by Panasonic Healthcare Co., Ltd.) was fitted into aconstant-pressure sample collection instrument (DU-AE01NT-H,manufactured by Panasonic Healthcare Co., Ltd.).[2] The swab was pressured with constant pressure against the monkeytongue, which was then scrubbed back and forth three times at intervalsof approximately 1 cm.1-3-5 Measurement of the Number of Bacteria in Oral Cavity by PlateCulture Technique

The agar plate pouring technique and the agar plate surface smearingtechnique were carried out with reference to New GMP Microbial TestingMethods and Standard Methods of Analysis in Food Safety Regulation.

[1] Each sampling solution into which the bacteria were recovered in1-3-3 was vigorously stirred, and the resultant was used as a recoveredbacterial suspension.

[2] 0.5 mL of the recovered bacterial suspension was diluted 10-fold,and dilution was further repeated by similar manipulation to make10-fold dilution series (5 scales).

[3] 1 mL each of the recovered bacterial suspension and the serialdilutions was dispensed to each dish. Approximately 15 mL of ameasurement medium (TSA+) preserved at approximately 47° C. was addedthereto to make pour plates. Also, 100 μL each of the recoveredbacterial suspension and the serial dilutions was dispensed to eachblood agar medium or each MS agar medium, and the surface was smearedusing a bacteria spreader.[4] After solidification of the measurement medium (TSA+), the pourplates were inverted, and cultured until colony counting was enabled.Also, the surface-smeared plates were inverted, and cultured underanaerobic conditions until colony counting was enabled.[5] Colonies that proliferated in the pour plates and thesurface-smeared plates were counted using a colony counter (DC-3, AS ONECorp.). A pour plate in which the number of colonies was too many todistinguish the colonies was regarded as TNTC (too numerous to count)without counting.1-3-6 Measurement of the Number of Bacteria in Oral Cavity UsingBacterial Counter[1] A bacterial counter (DU-AA01, manufactured by Panasonic HealthcareCo., Ltd.) was opened up.[2] A sensor chip of expendable supplies for measurement was fitted intothe bacterial counter.[3] A disposable cup of the expendable supplies for measurement wasloaded in the bacterial counter.[4] A swab into which bacteria were collected was loaded to the centerof the disposable cup.[5] The bacterial counter was closed.1-4 Results1-4-1 Measurement of the Number of Bacteria in Oral Cavity by PlateCulture Technique(1) Aerobic Culture

The results are shown in FIG. 1. The number of baseline bacteria in theoral cavity was 1.73×10⁵ to 4.20×10⁶. The number of bacteria in the oralcavity after saline embrocation was almost constant. The number ofviable bacteria was 6.48×10⁵ CFU, 4.65×10³ CFU and 2.35×10⁴ CFU 10minutes after test material embrocation, 1.66×10⁶ CFU, 1.47×10³ CFU and4.93×10⁵ CFU 6 hours after embrocation, and 4.67×10⁵ CFU, 5.58×10⁴ CFUand 1.22×10⁶ CFU 24 hours after embrocation, for saline, 0.1% OPB and0.47% PVP-I, respectively.

(2) Culture in Streptococcus Selective Medium

The results are shown in FIG. 2. The number of baseline bacteria in theoral cavity was 2.85×10⁵ to 7.60×10⁷. The number of bacteria in the oralcavity after saline embrocation was almost constant. The number ofviable bacteria was 1.26×10⁶ CFU, 5.97×10³ CFU and 7.33×10⁴ CFU 10minutes after test material embrocation, 5.51×10⁶ CFU, 2.79×10⁴ CFU and2.20×10⁶ CFU 6 hours after embrocation, and 1.71×10⁶ CFU, 4.30×10⁵ CFUand 7.81×10⁶ CFU 24 hours after embrocation, for saline, 0.1% OPB and0.47% PVP-I, respectively.

(3) Anaerobic Culture

The results are shown in FIG. 3. The number of baseline bacteria in theoral cavity was 2.45×10⁵ to 1.65×10⁷. The number of bacteria in the oralcavity after saline embrocation was almost constant. The number ofviable bacteria was 2.70×10⁶ CFU, 1.33×10⁴ CFU and 7.33×10⁴ CFU 10minutes after test material embrocation, 3.22×10⁶ CFU, 1.38×10⁴ CFU and1.80×10⁶ CFU 6 hours after embrocation, and 1.71×10⁶ CFU, 3.04×10⁵ CFUand 1.40×10⁶ CFU 24 hours after embrocation, for saline, 0.1% OPB and0.47% PVP-I, respectively.

1-4-2 Measurement of the Number of Bacteria in Oral Cavity UsingBacterial Counter

The results are shown in FIG. 4. The number of baseline bacteria in theoral cavity was 1.29×10⁶ to >1.00×10⁸. The number of viable bacteria was2.84×10⁶ CFU, <3.49×10⁵ CFU and 5.09×10⁵ CFU 10 minutes after testmaterial embrocation, 2.04×10⁷ CFU, 5.58×10⁵ CFU and 8.98×10⁶ CFU 6hours after embrocation, and 4.10×10⁷ CFU, 3.14×10⁷ CFU and 3.14×10⁷ CFU24 hours after embrocation, for saline, 0.1% OPB and 0.47% PVP-I,respectively.

Results having a similar tendency were obtained in both the measurementof the number of bacteria by the plate culture technique and themeasurement of the number of bacteria using a bacterial counter. Namely,the 0.1% OPB group kept the number of bacteria at a low value up to 6hours after embrocation, whereas the 0.47% PVP-I group merely exhibitedan effect up to 10 minutes after embrocation. However, the number ofbacteria made recovery 24 hours after embrocation in both the groups. Nopersistency was observed in the 0.47% PVP-I group probably because PVP-Iis susceptible to inactivation by organic matter in the oral cavity.0.1% OPB was considered to have a more persistent bactericidalantiseptic effect in the oral cavity and be superior therein.

Example 2

2. Bactericidal Test in Oral Cavity Using Hamster—1

This test was aimed at comparatively studying the bactericidal efficacyof a gargle (bactericidal antiseptic) on the mucosa in the oral cavityof normal hamsters with other agents. In order to study the persistencyof bactericidal activity, time points were established from after testmaterial gargling to 24 hours later (prior to, immediately after, 8hours after, and 24 hours after gargling), and the number of bacteriawas measured using a bacterial counter and the culture technique.

2-1 Test Material

Test substances and control substances were collectively used as testmaterials.

2-1-1 Test Substance 1

Designation: 0.1% OPB-1

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

2-1-2 Test Substance 2

Designation: 0.1% OPB-2

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

Lipidure® . . . 1.0 w/v %

2-1-3 Control Substance 1

Designation/abbreviated name: base/Base

Formula: Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

2-1-4 Control substance 2

Designation/abbreviated name: Peridex®/0.12% CHG

Formula: chlorhexidine gluconate . . . 0.12 w/v %

2-1-5 Control Substance 3

Designation/abbreviated name: Isodine Gargle Solution 0.47%/0.47% PVP-I

Formula: 15-fold dilution of Isodine Gargle Solution 7% (7% PVP-I,manufactured by Meiji Seika Pharma Co., Ltd.)

2-2 Animal Used

Male Slc: Syrian hamsters which were 6 weeks old upon receipt were usedto conduct a test on 4 animals per group.

2-3 Testing Method

2-3-1 Anesthesia

Gas anesthesia [induction of anesthesia: 3.0 L/min of air with 3%isoflurane (manufactured by Mylan Seiyaku Ltd.), the concentration ofcontinuous anesthesia was appropriately adjusted] was carried out.

2-3-2 Test Material Administration

Each hamster was fixed in the supine position under anesthesia, and 1 mLof each test material was injected to one cheek pouch. Thirty secondslater, the test material was eliminated, and a redundant test materialwas drawn out of the cheek pouch using a sterile swab.

2-3-3 Bacterial Collection

Bacteria were collected from both the cheek pouches under anesthesiausing a sterile swab at a total of 4 time points (before test materialadministration, 0 hr, 8 hr, and 24 hr). The swab after the collectionwas dipped in 5 mL of a SCDLP medium, then stirred, and used as a samplefor bacterial counting.

2-3-4 Measurement of the Number of Surviving Bacteria

The agar plate pouring technique was carried out with reference to NewGMP Microbial Testing Methods 1) and Standard Methods of Analysis inFood Safety Regulation 2).

[1] 500 μL of the sample for bacterial counting was collected, and10-fold dilution series from 10¹-fold to 10⁶-fold were made using 4.5 mLof a diluent solution.

[2] 1 mL each of the undiluted sample for bacterial counting and thediluted bacterial suspensions was dispensed to each sterile dish.

[3] 15 mL of a measurement medium (TSA+) incubated in a thermostat bathset to approximately 47° C. was rapidly dispensed to the dish.

[4] After solidification of the measurement medium, the resulting pourplates were inverted in an incubator, and cultured at 35° C. untilcolonies became able to be counted (approximately 2 days).

[5] After the culture, colonies that proliferated in the pour plateswere visually counted. A pour plate in which the number of colonies wastoo many to distinguish the colonies was regarded as TNTC (too numerousto count) without counting.

[6] The number of colonies was multiplied by the dilution ratio tocalculate the number of surviving bacteria.

2-4 Results

The results are shown in FIG. 5 and Table 2.

TABLE 2 The number of surviving bacteria in hamster oral cavity Thenumber of surviving bacteria {Mean ± SD[Log₁₀(CFU/swab)]} Test materialn Baseline 0 hr 8 hrs 24 hrs Base 4 6.09 ± 0.87 5.26 ± 0.50 5.55 ± 0.525.86 ± 0.53 0.1% OPB-1 4 6.13 ± 0.40 3.13 ± 0.52 3.98 ± 1.03 5.57 ± 0.690.1% OPB-2 4 6.16 ± 0.27 3.24 ± 0.34 4.52 ± 0.65 6.24 ± 0.22 Peridex 46.17 ± 0.65 4.04 ± 0.69 3.86 ± 0.92 5.68 ± 0.49 0.47% PVP-I 4 6.50 ±0.39 4.35 ± 0.33 5.79 ± 0.31 6.08 ± 0.47

The number of bacteria in the oral cavity before test materialadministration did not differ among the groups. The bactericidalefficacy was 0.1% OPB-1=0.1% OPB-2>0.12% CHG>0.47% PVP-I>Baseimmediately after test material administration, was 0.1% OPB-1=0.1%OPB-2=0.12% CHG>0.47% PVP-I=Base 8 hours after administration, and didnot differ among the groups 24 hours after administration. From theseresults, the bactericidal efficacy in the oral cavity was equivalentbetween 0.1% OPB and 0.12% CHG, and 0.47% PVP-I had a weak immediateeffect with no persistent activity observed.

The reason why the bactericidal activity of 0.47% PVP-I was low in thistest was that inactivation by proteins and the like in the oral cavityprobably made a significant contribution thereto. 0.12% CHG, as in 0.1%OPB, was considered as a bactericidal antiseptic having persistentactivity in the oral cavity.

Example 3

3. Bactericidal Test in Oral Cavity Using Hamster—2

This test was aimed at studying the influence of an olanexidineconcentration on the bactericidal efficacy of a gargle (bactericidalantiseptic) on the mucosa in the oral cavity of normal hamsters.

3-1 Test Material

Test substances and a control substance were collectively used as testmaterials.

3-1-1 Test Substance 1

Designation: 0.1% OPB-1

Formula: olanexidine gluconate . . . 0.10 w/v %

polyoxyethylene (20) polyoxypropylene (20) glycol . . . 0.07 w/v %

polyoxyethylene (160) polyoxypropylene (30) glycol . . . 0.10 w/v %

3-1-2 Test substance 2

Designation: 0.1% OPB-2

Formula: olanexidine gluconate . . . 0.10 w/v %

polyoxyethylene (20) polyoxypropylene (20) glycol . . . 0.07 w/v %

polyoxyethylene (160) polyoxypropylene (30) . . . 1.00 w/v %

3-1-3 Test Substance 3

Designation: 0.5% OPB-3

Formula: olanexidine gluconate . . . 0.50 w/v %

polyoxyethylene (20) polyoxypropylene (20) . . . 0.36 w/v %

polyoxyethylene (160) polyoxypropylene (30) . . . 5.00 w/v %

3-1-4 Test Substance 4

Designation: 1% OPB-2

Formula: olanexidine gluconate . . . 1.00 w/v %

polyoxyethylene (20) polyoxypropylene (20) glycol . . . 0.72 w/v %

polyoxyethylene (160) polyoxypropylene (30) glycol . . . 10.00 w/v %

3-1-5 Control Substance

Designation: base

Formula: polyoxyethylene (20) polyoxypropylene (20) glycol . . . 0.07w/v %

polyoxyethylene (160) polyoxypropylene (30) glycol . . . 0.10 w/v %

3-2 Animal Used

Male Slc: Syrian hamsters which were 6 weeks old upon receipt were usedto conduct a test on 3 animals per group.

3-3 Testing Method

3-3-1 Anesthesia

Gas anesthesia [induction of anesthesia: 3.0 L/min of air with 3%isoflurane (manufactured by Mylan Seiyaku Ltd.), the concentration ofcontinuous anesthesia was appropriately adjusted] was carried out.

3-3-2 Test Material Administration

Each hamster was fixed in the supine position under anesthesia, and 1 mLof each test material was injected to one cheek pouch. One minute later,the test material was eliminated, and a redundant test material wasdrawn out of the cheek pouch using a sterile swab.

3-3-3 Bacterial Collection

Bacteria were collected from both the cheek pouches under anesthesiausing a sterile swab at a total of 5 time points (before test materialadministration, 0 hr, 1 hr, 3 hr, and 6 hr). The swab after thecollection was dipped in 5 mL of a SCDLP medium, then stirred, and usedas a sample for bacterial counting.

3-3-4 Measurement of the Number of Surviving Bacteria

The number of surviving bacteria was measured in the same way as in2-3-4.

3-4 Results

The results are shown in FIG. 6. As is evident from the results, 0.1%OPB can reduce the number of bacteria to a low value persistently (up to6 hours later). The persistent activity was better at an OPBconcentration of 0.5 w/v % or higher.

Example 4

4. Oral Mucosal Irritancy Test Using Hamster—1

In this test, study drug formulations with varying base formulas of 0.1%olanexidine gluconate were repeatedly administered to the cheek pouchesof hamsters for 14 days, and comparatively studied for the degree ofirritancy.

4-1 Test Substance

4-1-1 Test substance 1

Designation: 0.1% OPB-1

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

4-1-2 Test Substance 2

Designation: 0.1% OPB-2

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic L-31 . . . 1.0 w/v %

4-1-3 Test Substance 3

Designation: 0.1% OPB-3

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

4-1-4 Test Substance 4

Designation: 0.1% OPB-4

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-85 . . . 1.0 w/v %

4-1-5 Test Substance 5

Designation: 0.1% OPB-5

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic F-127 . . . 1.0 w/v %

4-1-6 Test Substance 6

Designation: 0.1% OPB-6

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.14 w/v %

Pluronic F-68 . . . 1.0 w/v %

4-1-7 Test substance 7

Designation: 0.1% OPB-7

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Trehalose . . . 5.0 w/v %

4-2 Animal Used

Male Slc: Syrian hamsters which were 8 weeks old upon receipt were usedto conduct a test on 3 animals per group.

4-3 Testing Method

4-3-1 Test Substance Application Method

(1) Amount Applied

1 mL of each test substance was applied to the right cheek pouch.

(2) Application Method

[1] Anesthesia was induced by gas anesthesia [induction of anesthesia:3.0 L/min of air with 3% isoflurane (manufactured by Mylan SeiyakuLtd.)].

[2] Each animal was fixed in the supine position under maintenance ofanesthesia (the concentration was appropriately adjusted). The cheekpouch of the animal was pulled using a swab, and the pulled cheek pouchwas lightly pinched with one hand.

[3] Foreign matter such as feed attached to the mucosa of the cheekpouch was removed using saline and a swab for good hygiene. Then, thecheek pouch was put back in place.

[4] 1 mL of each test substance was applied to the right cheek pouchusing a 1 mL syringe and a probe for oral administration, and a vacantprobe for oral administration fitted into a 1 mL syringe was inserted tothe left cheek pouch, and decannulated.

[5] Thirty seconds after application, the animal was reversed to theprone position so as to prevent the backflow of the test substance intothe respiratory tract, and the test substance was eliminated. The wholeredundant test substance in the oral cavity was removed using a swab.[6] The color tone and the like of the cheek mucosa at the applicationsite were observed and recorded. A collar for hamsters was worn on theneck of the animal, and the animal was then brought back to a cage.[7] The manipulation described above was repeated twice a day (morningand evening) for 14 days.4-3-2 Examination and Observation(1) Observation of General Status

The general status was observed as to all the animals of each groupbefore application of the test material and at the completion ofapplication in the application period (Day 1 to Day 14). The observationwas also performed on the day following the end of the applicationperiod (Day 15).

(2) Body Weight Measurement

The body weight was measured as to all the animals of each group beforeapplication of the test material in the application period (Day 1 to Day14). The measurement was also performed on the day following the end ofthe application period (Day 15). However, the body weight was notmeasured on Days 13 and 14 due to the breakdown of a body weight scale.

(3) Macroscopic Observation Method at Application Site

The status of the mucosa of the cheek pouch was observed and scored asto the cheek pouches of all the animals of each group before applicationof the test material and at the completion of application in theapplication period (Day 1 to Day 14). The observation was also performedon the day (24±2 hours) following the end of the application period (Day15). The observation site was set to the cheek mucosa at a sitecontacted with each test material. As for the evaluation technique ofmacroscopic observation, the degrees of erythema and eschar formationwere numerically graded (stomatitis grade) according to the observationcriteria and the numerical grading described in Table 3 below (ISO10993-10, Annex B.3 “Table B.2 Grading system for oral and penilereactions”). Other detected manifestations were also recorded. On thebasis of the obtained observation results, the respective numericalgrades for the mucosa of the animals of each group were added for eachtest material, and the sum was divided by the number of observations andthe number of animals to determine an average value (rounded to unit),which was used as a reference material for comprehensive evaluation.

TABLE 3 Table B.2 Grading system for oral and penile reactions (Erythemaand eschar formation) Numerical grading No erythema 0 Very slighterythema (barely perceptible) 1 Well-defined erythema 2 Moderateerythema 3 Severe erythema (beet-redness) to eschar formation 4preventing grading of erythema(4) Pathological Examination

Each animal was sacrificed by blood-letting under isoflurane anesthesiaafter the completion of macroscopic observation on the day following theend of the application period, and the right and left cheek pouches werecollected and fixed in a 10% neutral buffered formalin solution.HE-stained specimens were made according to a routine technique, andpathological examination was carried out. As for the evaluationtechnique of macroscopic observation, manifestations or grades wererecorded as to each item of epithelium, leukocyte infiltration,hyperemia and edema according to the criteria described in ISO 10993-10,Annex B.3 “Table B.3 Grading system for microscopic examination fororal, penile, rectal and vaginal tissue reaction”. Other observedmanifestations were also recorded.

(5) Comprehensive Evaluation

The influence of each test material on the oral mucosa wascomprehensively evaluated on the basis of the degree of reaction of eachtest material obtained from the macroscopic observation results and thepathological observation results about the cheek mucosa, with referenceto transitions in general status and body weight in the observationperiod.

4-4 Results

4-4-1 General Status

No abnormality was observed in any of the animals.

4-4-2 Body Weight

The results are shown in FIG. 7. The body weight of the 0.1% OPB-5 groupwas hardly changed. The average values of the other groups weregradually increased.

4-4-3 Macroscopic Observation of Application Site

The results are shown in FIG. 8, and the cheek pouch of each group onthe final day is shown in Photos 1 to 8 of FIG. 9. Irritancy such aserythema was hardly observed in all the drug formulations. However, aleukoplakia-like symptom (increased keratosis or thickening) wasobserved in 0.1% OPB-1, -2, -6, -7 and -8. On the other hand, noabnormality was observed in 0.1% OPB-3, -4 and -5.

4-4-4 Histopathological Examination

The results are shown in FIG. 10. An average inflammation index of eachindividual and an average inflammation index of each group werecalculated by grading of epithelium (cell degeneration, metaplasia anderosion), leukocyte infiltration, hyperemia and edema according to theevaluation criteria described in ISO 10993-10, Annex B.3 “Table B.3Grading system for microscopic examination for oral, penile, rectal andvaginal tissue reaction”. Manifestations other than the evaluationcriteria were also recorded. As a result, no change was observed in theaverage value of the 0.1% OPB-3 group. Cell degeneration of theepithelium and minimum to moderate leukocyte infiltration were observedin the other groups including the 0.1% OPB-1 group, and the inflammationindex was evaluated as being the minimum of 1 to 3. In these groups,very slight intercellular edema and very slight to slight hyperkeratosiswere observed as manifestations other than the evaluation criteria.

These results suggested that the base Pluronic P-123 used for 0.1% OPB-3is particularly useful as a base for a drug formulation for applicationof olanexidine gluconate to the oral mucosa. The results also suggestedthat Pluronic P-85 and Pluronic F-127 used for 0.1% OPB-4 and -5, whichwere found to be free from abnormality in macroscopic observation, werealso usable as bases for a drug formulation for application ofolanexidine gluconate to the oral mucosa.

Example 5

5. Oral Mucosal Irritancy Test Using Hamster—2

The irritancy test of Example 4 suggested that the base Pluronic P-123is useful as a base for a drug formulation for application ofolanexidine gluconate to the oral mucosa. Accordingly, in this test,Pluronic P-123 was adopted as a base for making a drug formulationhaving no irritation, and subsequently, an OPB concentration and a baseconcentration were studied. The test system was carried out byperforming repeated administration to the hamster cheek pouch, andprolonging the period from 2 weeks to 4 weeks.

5-1 Test Substance

5-1-1 Test Substance 1

Designation: 0.1% OPB-1

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 0.50 w/v %

5-1-2 Test Substance 2

Designation: 0.1% OPB-2

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

5-1-3 Test Substance 3

Designation: 0.1% OPB-3

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 0.50 w/v %

Lipidure® . . . 1.0 w/v %

5-1-4 Test Substance 4

Designation: 0.1% OPB-4

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

Lipidure® . . . 1.0 w/v %

5-1-5 Test Substance 5

Designation: 0.3% OPB-1

Formula: olanexidine gluconate . . . 0.30 w/v %

Pluronic L-44 . . . 0.22 w/v %

Pluronic P-123 . . . 1.50 w/v %

5-1-6 Test Substance 6

Designation: 0.3% OPB-2

Formula: olanexidine gluconate . . . 0.30 w/v %

Pluronic L-44 . . . 0.22 w/v %

Pluronic P-123 . . . 3.0 w/v %

5-1-7 Test Substance 7

Designation: 0.3% OPB-3

Formula: olanexidine gluconate . . . 0.30 w/v %

Pluronic L-44 . . . 0.22 w/v %

Pluronic P-85 . . . 1.50 w/v %

5-1-8 Test Substance 8

Designation: 0.3% OPB-4

Formula: olanexidine gluconate . . . 0.30 w/v %

Pluronic L-44 . . . 0.22 w/v %

Pluronic P-85 . . . 3.0 w/v %

5-1-9 Test Substance 9

Designation: 0.5% OPB-1

Formula: olanexidine gluconate . . . 0.50 w/v %

Pluronic L-44 . . . 0.36 w/v %

Pluronic P-123 . . . 2.50 w/v %

5-1-10 Test Substance 10

Designation: 0.5% OPB-2

Formula: olanexidine gluconate . . . 0.50 w/v %

Pluronic L-44 . . . 0.36 w/v %

Pluronic P-123 . . . 5.0 w/v %

5-1-11 Test Substance 11

Designation: 0.5% OPB-3

Formula: olanexidine gluconate . . . 0.50 w/v %

Pluronic L-44 . . . 0.36 w/v %

Pluronic P-123 . . . 2.50 w/v % Lipidure® . . . 1.0 w/v %

5-1-12 Test Substance 12

Designation: 0.5% OPB-4

Formula: olanexidine gluconate . . . 0.50 w/v %

Pluronic L-44 . . . 0.36 w/v %

Pluronic P-123 . . . 5.0 w/v %

Lipidure® . . . 1.0 w/v %

5-2 Animal Used

Male Slc: Syrian hamsters which were 8 weeks old upon receipt were usedto conduct a test on 3 animals per group.

5-3 Testing Method

5-3-1 Test Substance Application Method

(1) Amount Applied

1 mL of each test substance was applied to the left cheek pouch.

(2) Application Method

[1] Anesthesia was induced by gas anesthesia [induction of anesthesia:3.0 L/min of air with 3% isoflurane (manufactured by Mylan SeiyakuLtd.)].

[2] Each animal was fixed in the supine position under maintenance ofanesthesia (the concentration was appropriately adjusted). The cheekpouch of the animal was pulled using a swab, and the pulled cheek pouchwas lightly pinched with one hand.

[3] Foreign matter such as feed attached to the mucosa of the cheekpouch was removed using saline and a swab for good hygiene. Then, thecheek pouch was put back in place.

[4] 1 mL of each test substance was applied to the left cheek pouchusing a 1 mL syringe and a probe for oral administration, and a vacantprobe for oral administration fitted into a 1 mL syringe was inserted tothe right cheek pouch, and decannulated.

[5] Thirty seconds after application, the animal was reversed to theprone position so as to prevent the backflow of the test substance intothe respiratory tract, and the test substance was eliminated. The wholeredundant test substance in the oral cavity was removed using a swab.[6] The color tone and the like of the cheek mucosa at the applicationsite were observed and recorded, and the animal was then brought back toa cage.[7] The manipulation described above was repeated twice a day (morningand evening) for 28 days.5-3-2 Examination and Observation(1) Observation of General Status

The general status was observed as to all the animals of each groupbefore application of the test material and at the completion ofapplication in the application period (Day 1 to Day 28). The observationwas also performed on the day following the end of the applicationperiod (Day 29).

(2) Body Weight Measurement

The body weight was measured as to all the animals of each group beforeapplication of the test material in the application period (Day 1 to Day28). The measurement was also performed on the day following the end ofthe application period (Day 29).

(3) Macroscopic Observation Method at Application Site

The status of the mucosa of the cheek pouch was observed and scored asto the cheek pouches of all the animals of each group before applicationof the test material in the application period (Day 1 to Day 28). Theobservation was also performed on the day (24±2 hours) following the endof the application period (Day 29). The observation site was set to thecheek mucosa at a site contacted with each test material. As for theevaluation technique of macroscopic observation, the degrees of erythemaand eschar formation were numerically graded (stomatitis grade)according to the observation criteria and the numerical gradingdescribed in Table 3 above (ISO 10993-10, Annex B.3 “Table B.2 Gradingsystem for oral and penile reactions”). Other detected manifestationswere also recorded. On the basis of the obtained observation results,the respective numerical grades for the mucosa of the animals of eachgroup were added for each test material, and the sum was divided by thenumber of observations and the number of animals to determine an averagevalue (rounded to unit), which was used as a reference material forcomprehensive evaluation.

(4) Pathological Examination

Each animal was sacrificed by blood-letting under isoflurane anesthesiaafter the completion of macroscopic observation on the day following theend of the application period, and the right and left cheek pouches werecollected and fixed in a 10% neutral buffered formalin solution.HE-stained specimens were made according to a routine technique, andpathological examination was carried out. As for the evaluationtechnique of macroscopic observation, manifestations or grades wererecorded as to each item of epithelium, leukocyte infiltration,hyperemia and edema according to the criteria described in ISO 10993-10,Annex B.3 “Table B.3 Grading system for microscopic examination fororal, penile, rectal and vaginal tissue reaction”. Other observedmanifestations were also recorded.

(5) Comprehensive Evaluation

The influence of each test material on the oral mucosa wascomprehensively evaluated on the basis of the degree of reaction of eachtest material obtained from the macroscopic observation results and thepathological observation results about the cheek mucosa, with referenceto transitions in general status and body weight in the observationperiod.

5-4 Results

5-4-1 General Status

No abnormality was observed in any of the animals.

5-4-2 Body Weight

The body weight was increased over time in all the groups, and hardlydiffered among the groups.

5-4-3 Macroscopic Observation of Application Site

The results are shown in FIG. 11. Irritancy such as erythema was notobserved in all the drug formulations (numerical grading: 0). However, aleukoplakia-like symptom (increased keratosis or thickening) wasobserved in OPB having a concentration of 0.3% or higher. On the otherhand, no abnormality was observed in OPB having a concentration of 0.1%.

5-4-4 Histopathological Examination

The results are shown in FIG. 12. An average inflammation index of eachindividual and an average inflammation index of each group werecalculated by grading of epithelium (cell degeneration, metaplasia anderosion), leukocyte infiltration, hyperemia and edema according to theevaluation criteria described in ISO 10993-10, Annex B.3 “Table B.3Grading system for microscopic examination for oral, penile, rectal andvaginal tissue reaction”. Manifestations other than the evaluationcriteria were also recorded. As a result, no inflammatory reaction wasobserved in each group of 0.1% OPB. Degeneration of the epithelium andleukocyte infiltration were observed in each group of OPB having aconcentration of 0.3% or higher, and all the reactions were minimal withan inflammation index of 1 to 3. Very slight to slight hyperkeratosiswas observed as manifestations other than the evaluation criteria insome individuals of the 0.1% OPB group, and very slight to moderatehyperkeratosis and very slight outgrowth of prickle cells were observedin each group of OPB having a concentration of 0.3% or higher. Inaddition, intraepidermal microabscess observed in the control group(right cheek pouch: Sham-ope side) seemed to be a naturally occurringlesion.

Example 6

6. Efficacy Test in 5-FU-Induced Hamster Stomatitis Model—1

In this test, the efficacy of an OPB drug formulation was tested in5-FU-induced stomatitis models. Specifically, measurements of the numberof bacteria in the oral cavity over time and stomatitis evaluation wereperformed by gargling in the oral cavity with 0.1% OPB in 5-FU-inducedstomatitis models.

6-1 Test Material

A test substance and a control substance were collectively used as testmaterials.

6-1-1 Test Substance

Designation: 0.1% OPB

Formula: olanexidine gluconate . . . 0.10 w/v %

polyoxyethylene (20) polyoxypropylene (20) glycol . . . 0.14 w/v %

polyoxyethylene (160) polyoxypropylene (30) glycol . . . 0.10 w/v %

6-1-2 Control Substance

Designation: base

Formula: polyoxyethylene (20) polyoxypropylene (20) glycol . . . 0.07w/v %

polyoxyethylene (160) polyoxypropylene (30) glycol . . . 0.10 w/v %

6-2 Animal Used

Male Slc: Syrian hamsters which were 6 weeks old upon receipt were usedto conduct a test on 5 animals per group.

6-3 Testing Method

6-3-1 Anesthesia

Gas anesthesia [induction of anesthesia: 3.0 L/min of air with 3%isoflurane (manufactured by Mylan Seiyaku Ltd.), the concentration ofcontinuous anesthesia was appropriately adjusted] was carried out.

6-3-2 Stomatitis Model Making

5-FU was intraperitoneally administered at 60 mg/kg to the hamstersunder anesthesia. The administration was performed a total of twice onDay 0 and Day 2.

On Day 4, the cheek pouch was pulled out from each hamster underanesthesia. Feed and floor mat for laboratory animals accumulated in thecheek pouch were removed, and the cheek pouch was patted with a cottonpad saturated with saline. The surface layer (horny layer) of the cheekpouch was brushed with a precision wire brush (ϕ2.34 mm, manufactured bySumflex. Co., Ltd.). The cheek pouch thus brushed was brought back tothe oral cavity.

6-3-3 Test Material Administration

Each hamster was fixed in the supine position under anesthesia, and 1 mLof each test material was injected to one cheek pouch. Thirty secondslater, the test material was eliminated, and a redundant test materialwas drawn out of the cheek pouch using a sterile swab. Thisadministration by the gargling manipulation was performed twice a day.The administration was not carried out after the disorder of stomatitisreached the peak.

6-3-4 Bacterial Collection

On Days 0, 4, 7, 10, and 17, bacteria were collected from both the cheekpouches under anesthesia using a sterile swab at a total of 4 timepoints (before the first test material administration, 0 hr, and 6 hrlater). However, on days 10 and 17 without test material application,bacteria were collected only once. The swab after the collection wasdipped in 5 mL of a SCDLP medium, then stirred, and used as a sample forbacterial counting.

6-3-5 Measurement of the Number of Surviving Bacteria

The agar plate pouring technique was carried out with reference to NewGMP Microbial Testing Methods 1) and Standard Methods of Analysis inFood Safety Regulation 2).

[1] 500 μL of the sample for bacterial counting was collected, and10-fold dilution series from 10¹-fold to 10⁴-fold were made using 4.5 mLof a diluent solution.

[2] 1 mL each of the undiluted sample for bacterial counting and thediluted bacterial suspensions was dispensed to each sterile dish.

[3] 15 mL of a measurement medium (TSA+) incubated in a thermostat bathset to approximately 47° C. was rapidly dispensed to the dish.

[4] After solidification of the measurement medium, the resulting pourplates were inverted in an incubator, and cultured at 35° C. untilcolonies became able to be counted (approximately 2 days).

[5] After the culture, colonies that proliferated in the pour plateswere visually counted. A pour plate in which the number of colonies wastoo many to distinguish the colonies was regarded as TNTC (too numerousto count) without counting.

6-3-6 Calculation of the Number of Surviving Bacteria

The number of colonies adopted on the basis of the section 6-3-5 wasdivided by the dilution ratio to determine the number of survivingbacteria (CFU/mL). The number of colonies adopted was rounded off to onedecimal place and displayed. The number of surviving bacteria (CFU/swab)was calculated according to the following expression.

A: the number of colonies adoptedThe number of surviving bacteria (CFU/swab)=A×Dilution ratio×Amount ofthe sample fluid (5 mL)

Log reduction was further determined according to the expression givenbelow from the logarithmic value of the number of surviving bacteria.The log reduction was rounded off to two decimal places and displayed.When the number of surviving bacteria was 1 or less, the logarithmicvalue was set to 0.

B: logarithmic value of the number of viable bacteria at the baseline

C: logarithmic value of the number of viable bacteria after testmaterial embrocationLog reduction=B−C6-3-7 Statistical Analysis

A mean and standard deviation were determined on the number of viablebacteria (CFU/swab) of each group and its logarithmic value. The numberof viable bacteria was rounded to unit and displayed in integer. Thelogarithmic value of the number of viable bacteria was rounded off totwo decimal places and displayed. When the number of viable bacteria was0, the logarithmic value of the number of viable bacteria was set to 0.No assay was conducted because of an exploratory test.

6-3-8 Stomatitis Evaluation

A stomatitis grade was evaluated on the basis of Table 4 below.

TABLE 4 Grade Status 0 Neither erythema nor vasodilation 1 Erythema andvasodilation 2 Serious erythema attended with superficial mucosalerosion 3 Mucosal ulceration (25%) 4 Mucosal ulceration (50%) 5 Mucosalulceration (100%)6-4 Results6-4-1 the Number of Bacteria

The results are shown in Table 5 and FIG. 13. Decrease in the number ofbacteria after administration was marked in the 0.1% OPB group on Days0, 4, and 10, whereas the value of the decreased number of bacteria wasvery small on Day 7 when increase in severity of stomatitis was marked.

TABLE 5 The number of surviving bacteria in hamster oral cavity Thenumber of surviving bacteria {Mean ± SD [Log₁₀ (CFU/swab)]} Test 0 day 4day 7 day 10 day 17 day material n pre 0 h 6 h pre 0 h 6 h pre 0 h 6 hPre 0 h 6 h pre No 5 6.78 ± 6.72 ± 6.79 ± 7.07 ± 6.97 ± 6.82 ± 7.03 ±6.84 ± 6.90 ± 6.59 ± 6.54 ± 6.54 ± 6.65 ± procedure 0.43 0.38 0.22 0.490.53 0.20 0.48 0.35 0.21 0.26 0.30 0.18 0.16 Base 5 6.61 ± 5.95 ± 6.10±6.62 ± 6.08 ± 6.30 ± 7.12 ± 6.89 ± 6.68 ± 6.61 ± 6.67 ± 6.85 ± 6.68 ±0.52 0.39 0.39 0.42 0.09 0.20 0.07 0.29 0.23 0.59 0.57 0.31 0.56 0.1%OPB 5 6.86 ± 4.69 ± 4.78 ± 6.78 ± 5.32 ± 6.31 ± 7.02 ± 6.36 ± 6.84 ±6.60 ± 5.82 ± 6.54 ± 6.50 ± 0.42 0.15 0.68 0.30 0.25 0.55 0.19 0.44 0.350.35 0.27 0.32 0.276-4-2 Stomatitis Grade

The results are shown in FIG. 14. The stomatitis grade was markedly lowin the 0.1% OPB group.

In this test, the value of the decreased number of bacteria was low onDay 7 in the 0.1% OPB group probably because of reduction inbactericidal activity due to the bacterial collection method or anexcess of an effusion. This test suggested that increase in severity ofstomatitis is mitigated by administering 0.1% OPB and thereby keepingthe oral cavity clean.

Example 7

7. Efficacy Test in 5-FU-Induced Hamster Stomatitis Model—2

In this test, the stomatitis-mitigating effects of 0.1% olanexidinegluconate and 0.1% CHG were comparatively studied in 5-FU-inducedhamster stomatitis models.

7-1 Test Material

Test substances and a control substance were collectively used as testmaterials.

7-1-1 Test Substance 1

Designation: 0.1% OPB

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

7-1-2 Test Substance 2

Designation: Peridex®/0.1% CHG

Manufacturer: 3M ESPE Dental Products

Formula: chlorhexidine gluconate . . . 0.12 w/v %

7-1-3 Control substance

Designation: base

Formula: polyoxyethylene (20) polyoxypropylene (20) glycol . . . 0.07w/v %

7-2 Bacterium Used

In this test, Staphylococcus aureus (ATCC No: 6538, manufactured byMicrobiologics, Inc.) was used, which is a normal inhabitant in the oralcavity.

7-3 Animal Used

Male Slc: Syrian hamsters which were 6 weeks old upon receipt were usedto conduct a test on 5 animals per group.

7-4 Testing Method

7-4-1 Preparation of Test Bacterial Suspension

[1] A stored vial containing bacterial pellets was taken out and broughtback to room temperature.

[2] One bacterial pellet was taken out of the vial and transferred to asterile tube.

[3] 0.5 mL of saline was added thereto.

[4] The bacterial pellet was squashed with a sterile swab to prepare asuspended bacterial fluid.

[5] The suspended bacterial fluid was inoculated to a round area ofapproximately 2 cm in diameter in a TSA plate using a sterile swab, andstreaked from the inoculation area using a platinum loop.

[6] The streaked TSA plate was inverted, and cultured until colonieswere formed.

[7] A single colony was selected from among the formed colonies,collected with a platinum needle, and stabbed to a Casitone medium.

[8] The Casitone medium in which the inoculant was stabbed was cultureduntil proliferation of bacteria became able to be confirmed.

[9] After confirmation of the proliferation of bacteria, the bacteriawere refrigerated (set value: 2 to 8° C.).

[10] A portion of the test bacteria refrigerated in the Casitone mediumwas collected with a platinum needle, transferred to a 14 mL steriletube containing 5 mL of an MHB medium, and static cultured until thebacteria proliferated.

[11] After the culture, 10 μL of the culture solution was collected witha sterile tip, transferred again to a 14 mL sterile tube containing 5 mLof an MHB medium, and static cultured until the bacteria proliferated.

[12] After the culture, approximately 5 mL of the test bacterial culturesolution subcultured in the MHB medium was recovered into a 15 mLconical tube. After addition of 8 mL of saline, the mixture was gentlystirred.

[13] The tube was centrifuged at 3000 rpm at 23° C. for 10 minutes(cooled centrifuge 5800, rotor RS-720, manufactured by Kubota Corp.),and the supernatant was discarded.

[14] The precipitated test bacteria were suspended by the addition of 1mL of distilled water (Otsuka Distilled Water, manufactured by OtsukaPharmaceutical Factory, Inc.).

[15] The suspended bacterial fluid was transferred to a 14 mL steriletube, and the turbidity was determined using McFarland Standard (productNo. 70900, manufactured by Sysmex-Biomerieux Co., Ltd.”). Theconcentration of the bacterial suspension was adjusted by the additionof saline so as to attain McFarland 5.[16] The suspended bacterial fluid adjusted to McFarland 5 was used as atest bacterial suspension.7-4-2 Anesthesia

Gas anesthesia [induction of anesthesia: 3.0 L/min of air with 3%isoflurane (Mylan Seiyaku Ltd.), the concentration of continuousanesthesia was appropriately adjusted] was carried out.

7-4-3 Stomatitis Model Making

5-FU was intraperitoneally administered at 60 mg/kg to the hamstersunder anesthesia. The administration was performed a total of twice onDay 0 and Day 2. On Day 4, the cheek pouch was pulled out from eachhamster under anesthesia. Feed and floor mat for laboratory animalsaccumulated in the cheek pouch were removed, and the cheek pouch waspatted with a cotton pad saturated with saline. The surface layer (hornylayer) of the cheek pouch was brushed with a precision wire brush (ϕ2.34mm, manufactured by Sumflex. Co., Ltd.). The cheek pouch thus brushedwas brought back to the oral cavity.

7-4-4 Test Material Administration

Each test material was embrocated twice a day to the hamster cheek pouchunder anesthesia using a swab (for 4 days from the grouping day).However, on the 4th day, the administration was performed once (only inthe morning).

7-4-5 Test Bacterial Suspension Embrocation

The test bacterial suspension prepared in 7-4-1 was embrocated once aday before the test material administration in the morning to thehamster cheek pouch under anesthesia using a platinum loop (for 5 daysfrom the grouping day).

7-4-6 Stomatitis Evaluation

A stomatitis grade was evaluated on the basis of Table 4 above.

7-4-7 Statistical Analysis

A mean and standard deviation were determined on the grade of eachgroup, and a graph was created using only the mean. No assay wasconducted because of exploratory analysis.

7-5 Results

The results are shown in FIG. 15. A tendency to mitigate increase inseverity of stomatitis was seen in the 0.1% OPB group compared with thebase and 0.1% CHG groups. The grade was almost the same with nodifference between the base group and the 0.1% CHG group. From theeffect of mitigating increase in severity of stomatitis, the 0.1% OPBwas considered to be superior in bactericidal efficacy on the embrocatedbacteria or the normal inhabitant in the mucosa of the cheek pouch to0.1% CHG.

Example 8

8. Efficacy test in hamster model of stomatitis induced by using 5-FUand radiation irradiation in combination

In this test, a drug formulation of 0.1% OPB containing Pluronic P-123as a base was applied by the gargling technique to hamster models ofstomatitis induced by using 5-FU and radiation irradiation incombination, and comparatively studied for a stomatitis-mitigatingeffect.

8-1 Test material

A test substance and a control substance were collectively used as testmaterials.

8-1-1 Test substance

Designation: OPB

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

8-1-2 Control substance

Designation/abbreviated name: base/Base

Formula: Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

8-2 Animal Used

Male Slc: Syrian hamsters which were 6 weeks old upon receipt were usedto conduct a test on 8 animals per group.

8-3 Testing Method

8-3-1 Anesthesia

(1) At Time of Radiation Irradiation

Somnopentyl® (manufactured by Kyoritsuseiyaku Corp.) wasintraperitoneally administered at 40 mg/kg.

(2) At time of stomatitis evaluation and test material administration

Gas anesthesia [induction of anesthesia: 3.0 L/min of air with 3%isoflurane (manufactured by Mylan Seiyaku Ltd.), the concentration ofcontinuous anesthesia was appropriately adjusted] was carried out.

8-3-2 Stomatitis Model Making

(1) Radiation irradiation

On Day 0, the cheek pouch was pulled out from each hamster underanesthesia using a swab. Feed and floor mat for laboratory animalsaccumulated in the cheek pouch were removed, and the cheek pouch waspatted with a cotton pad saturated with saline. The body and the cheekpouch were both fixed onto a molded acrylic plate. A region other thanthe cheek pouch at an irradiation site was covered with lead, and thecheek pouch was irradiated with radiation (40 Gy) under conditions of[shelf board distance: 12.5 cm, tube voltage: 160 kV, tube current: 6.2mA]. However, the irradiation was performed for one cheek pouch perindividual, and four animals with the left cheek pouch irradiated andfour animals with the right cheek pouch irradiated were assigned to eachgroup.

(2) 5-FU administration

5-FU was intraperitoneally administered at 60 mg/kg to the hamsters atotal of three times on Days 0, 5, and 10.

8-4-3 Test material administration

Each hamster was fixed in the supine position under anesthesia, and 1 mLof each test material was injected to one cheek pouch. Thirty secondslater, the test material was eliminated, and a redundant test materialwas drawn out of the cheek pouch using a sterile swab. Thisadministration by the gargling manipulation was performed twice a day.The administration was not carried out after the disorder of stomatitisreached the peak.

8-4-4 Stomatitis Evaluation

A stomatitis grade was evaluated on the basis of Table 4 above.

8-4-5 Statistical Analysis

A mean and standard deviation were determined on the stomatitis grade ofeach group, and a graph was created. No assay was conducted because ofexploratory analysis.

8-5 Results

The results are shown in FIG. 16. The maximum value of the stomatitisgrade was 4.9 for the base group and 4.3 for the OPB group, andfurthermore, the grade started to rise earlier in the base group. TheOPB group was evidently cured earlier, though all the animals were notcompletely cured because ulcer in some individuals was not healed evenon Day 40 due to the influence of large strength of the models.

Example 9

9. Efficacy Test in Rat Gingivitis Model

In this test, the therapeutic effect of 0.1% olanexidine gluconate ongingivitis was studied in rat gingivitis models.

9-1 Test Material

A test substance and a control substance were collectively used as testmaterials.

9-1-1 Test substance

Designation: OPB

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

9-1-2 Control substance

Designation/abbreviated name: base/Base

Formula: Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

9-2 Bacterium Used

In this test, Porphyromonas gingivalis (ATCC No: 33277, manufactured byMicrobiologics, Inc.) was used, which is a bacterium causative ofgingivitis.

9-3 Animal Used

Male Jcl: Wistar rats which were 5 weeks old upon receipt were used toconduct a test on 5 animals per group.

9-4 Testing Method

9-4-1 Preparation of Test Bacterium (the Whole Manipulation Except forPreservation was Performed in an Anaerobic Chamber)

[1] A stored vial containing bacterial pellets was taken out and placedin an anaerobic chamber.

[2] One bacterial pellet was taken out of the vial and transferred to asterile tube.

[3] 0.5 mL of a prepared TSB medium was added thereto.

[4] The bacterial pellet was squashed with a sterile swab to prepare asuspended bacterial fluid.

[5] The suspended bacterial fluid was inoculated to a sheep blood agarmedium for CDC anaerobes.

[6] Culture was performed for 3 to 4 days under anaerobic conditions(37° C.)

[7] A single colony was selected from among the formed colonies, andsimilarly inoculated again to a sheep blood agar medium for CDCanaerobes.

[8] After confirmation of proliferation of bacteria, 3 mL of a preparedTSB medium was added thereto, and the bacteria were suspended using aspreader to make a glycerol stock.

[9] The stock was cryopreserved.

[10] The stock was inoculated to a sheep blood agar medium for CDCanaerobes and cultured under anaerobic conditions.

[11] After confirmation of proliferation of bacteria, the bacteria weresuspended in an appropriate amount of a prepared TSB medium. A portionof the suspension was taken out, and the turbidity was adjusted toMcFarland 5 using McFarland Standard. The dilution ratio was calculated,and the concentration of the bacterial suspension was adjusted to 1×10¹⁰CFU/mL from the remaining suspension.[12] The resultant was used as a test bacterial suspension.9-4-2 Anesthesia(1) At time of cotton sewing thread insertion and autopsy

An aqueous pentobarbital sodium solution was intraperitoneallyadministered at 40 mg/kg.

(2) Bacterial inoculation and test material administration

Gas anesthesia [induction of anesthesia: 1.0 L/min of air with 5%isoflurane (Mylan Seiyaku Ltd.), the concentration of continuousanesthesia was appropriately adjusted] was carried out.

9-4-3 Cotton sewing thread insertion

After anesthesia, each animal was fixed in the dorsal position to adedicated table, and a cotton sewing thread was inserted to between theupper right first and second molars with the lower jaw lifted.

9-4-4 Bacterial Inoculation

After anesthesia, 0.2 mL of the test bacterial suspension was inoculatedto the cotton sewing thread insertion site. This manipulation wascarried out every 2 hours.

9-4-5 Test material administration

After anesthesia, 1 mL of each test material was administered to cleansethe oral cavity. This manipulation was performed twice a day.

9-4-6 Observation and examination

(1) General Status

The general status was observed once a day from the cotton sewing threadinsertion day to the autopsy day.

(2) Body Weight Measurement

The body weight was measured a total of twice from the cotton sewingthread insertion day to the autopsy day.

(3) Autopsy

Each animal was sacrificed by blood-letting from the cut abdominal aortaunder anesthesia, and autopsy was performed.

(4) Histopathological Examination

The excised upper jaw was fixed in a 10 v/v % neutral buffered formalinsolution, degreased, and decalcified, and HE-stained specimens were thenmade. Inflammatory change was pathologically examined as to eachspecimen.

9-4-7 Statistical analysis

A mean and standard deviation were calculated on the body weight of eachgroup. No assay was conducted.

9-5 Results

No abnormality was observed in the general status, and the body weightdid not differ between the groups.

The pathological examination results are shown in FIG. 17. Themicrographs of the HE-stained specimens are shown in FIG. 18.

In the OPB administration group, the stratified squamous epithelium ofthe gingiva was observed with infiltration of neutrophils in 8 out of 10cases, intercellular edematization in 1 out of 10 cases, and ulcer in 1out of 10 cases, all of which were very slight. The lamina propria ofthe gingiva was observed with infiltration of neutrophils in 8 out of 10cases and bleeding in 1 out of 10 cases, all of which were very slight.On the other hand, in the base administration group, the stratifiedsquamous epithelium of the gingiva was observed with infiltration ofneutrophils which was very slight in 8 out of 10 cases and was slight in2 out of 10 cases. The stratified squamous epithelium of the gingiva wasobserved with intercellular edematization in 2 out of 10 cases,hyperkeratosis in 2 out of 10 cases, acanthosis in 2 out of 10 cases andulcer in 1 out of 10 cases, all of which were very slight. The laminapropria of the gingiva was observed with infiltration of neutrophilswhich was very slight in 6 out of 10 cases and was slight in 3 out of 10cases. The lamina propria of the gingiva was observed with bleeding andedematization, both of which were very slight in 1 out of 10 cases.

9-6 Discussion

All of infiltration of neutrophils, intercellular edematization,hyperkeratosis and acanthosis in the stratified squamous epithelium ofthe gingiva, and infiltration of neutrophils and edematization in thelamina propria were changes associated with an inflammation, and wereconsidered to occur due to procedures. All of these changes tended to below in terms of both frequency and degree in the OPB administrationgroup compared with the base administration group. Therefore, aninflammation-mitigating effect brought about by OPB administration wasobserved.

Example 10

10. Efficacy Test in Rat Pneumonia Model

In this test, the therapeutic effect of 0.1% olanexidine gluconate onpneumonia was studied in rat aspiration pneumonia models.

10-1 Test Material

A test substance and a control substance were collectively used as testmaterials.

10-1-1 Test Substance

Designation: OPB

Formula: olanexidine gluconate . . . 0.10 w/v %

Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

10-1-2 Control substance

Designation/abbreviated name: base/Base

Formula: Pluronic L-44 . . . 0.07 w/v %

Pluronic P-123 . . . 1.0 w/v %

10-2 Animal Used

Male Crl: CD (SD) rats which were 7 weeks old upon receipt were used toconduct a test.

10-3 Group Configuration

The body weight was measured in the morning on the intratrachealadministration day, and the animals were assigned by stratifiedrandomization to 4 groups (groups 1 to 4) shown in Table 6 below. Sixanimals excluded from the assignment were assigned by stratifiedrandomization to 2 groups (groups 5 and 6) shown in Table 6 below, andused as individuals for saliva collection.

TABLE 6 Timing of BALF collection Group Administered (elapsed time afterintratracheal No. substance administration) n 1 Saliva (collected  6 h 6from group 5) 2 Saliva (collected 24 h 6 from group 5) 3 Saliva(collected  6 h 6 from group 5) 4 Saliva (collected 24 h 6 from group 5)Individual for BALF recovery Group Oral cavity cleansing No. (testmaterial) n 5 OPB 3 6 Base 3 Individual for saliva collection10-4 Testing method10-4-1 Anesthesia(1) Cleansing of Oral Cavity and Saliva Collection

Somnopentyl was intraperitoneally administered at 40 mg/kg.

(2) At Time of Intratracheal Administration and Bronchoalveolar LavageFluid (BALF) Collection

Gas anesthesia [induction of anesthesia: 3.0 L/min of air with 3%isoflurane (Mylan Seiyaku Ltd.), the concentration of continuousanesthesia was appropriately adjusted] was carried out.

10-4-2 Cleansing of Oral Cavity

A sterile swab was impregnated with each test material, which was thenembrocated in a sufficient amount to the oral cavity under anesthesia.

10-4-3 Saliva collection

0.1% pilocarpine hydrochloride (5 mg/kg) was intraperitoneallyadministered under anesthesia. Over-secreted saliva was recovered. Therecovered saliva was added to a nutritive medium containing aneutralizing agent, and left standing. Then, centrifugation (r.t., 3000rpm, 10 min) was performed, and sediments were suspended in the sameamount of saline to prepare an intratracheal administration solution.

10-4-4 Intratracheal Administration

After saliva collection, a tube for administration was indwelled in thetrachea under anesthesia using a pharyngoscope, and 0.1 mL of saliva orsaline was administered thereto.

10-4-5 BALF Collection

A median incision was made under anesthesia 6 or 24 hours afterintratracheal administration, and each animal was euthanized byblood-letting from the incision in the abdominal aorta. Then, the lungwas exposed, and a catheter was inserted to the origin of the bronchus.Lavage was performed three times (infusion and recovery were repeatedtwice for each time) with 8 mL of a PBS solution containing 0.1% BSA and0.05 mM EDTA-2Na (hereinafter, referred to as PBS) through the line, anda lavage fluid was collected (BALF). BALF was centrifuged (200 g, 4° C.,10 min), and the supernatant was separated into other preservation tubesand used for LDH concentration measurement and measurement of cytokines(ELISA). Sediments were suspended in 1 mL of PBS and used forhematological examination.

10-4-6 Handling of Animal for Saliva Collection

After the completion of intratracheal administration, each animal forsaliva collection was euthanized by blood-letting under, excessanesthesia.

10-4-7 Cytokine (TNF-α and IL-6) Concentration Measurement

The measurement was performed according to protocols included in kits.

10-4-8 Hematological Examination

Hematological analysis was carried out on the suspended sediments usingan automatic blood cell counter for multiple items.

10-4-9 Biochemical Examination

An LDH concentration in the collected BALF supernatant was measuredusing an automatic analysis apparatus 7180 (Hitachi High-TechnologiesCorp.).

10-4-10 Statistical Analysis

No assay was conducted because of exploratory analysis.

10-5 Results

The hematological examination and biochemical examination results areshown in FIG. 19.

There was no difference between both the groups in the hematologicalexamination. The 24-hour value of IL-6 was lower by 2.5 times in the OPBgroup. The value of TNF-α was lower in the OPB group at both the timepoints.

These results suggested that inflammatory reaction in the lung due toaspiration of saliva is lower in dealing of the oral cavity with OPB.

Example 11

11. Study on Anti-Inflammatory Action of Olanexidine Using TLR ReporterCell Line

Examples 6 to 10 indicated that olanexidine has anti-inflammatory actionon stomatitis, gingivitis, and pneumonia. It has been revealed thatinflammations are associated with immune response mediated by Toll-likereceptor 4 (TLR-4) and Toll-like receptor 2 (TLR-2), which are receptorsrecognizing LPS or LTA (ChemMedChem. 2016 January 19; 11 (2): 154-65;Biotechnol Adv. 2012 January-February; 30 (1): 251-60; and J Dent Res.2016 July; 95 (7): 725-33).

Olanexidine has the possibility of suppressing an inflammation byantagonistic (antagonist-like) action on TLR-4 and TLR-2. Accordingly,in this test, in order to elucidate this, the antagonistic(antagonist-like) action of olanexidine on TLR-4 and TLR-2 was confirmedby reporter assay using human-derived cells stably expressing TLR-4,TLR-2, and reporter (SEAP) genes.

11-1 Test substance

Designation: 1.5% OPB

Formula: olanexidine gluconate . . . 1.5 w/v %

11-2 Cell

The cells described in Table 7 below were used.

TABLE 7 Designation Catalog (abbreviated name) Host cell Expressed geneNo./supplier Medium TLR4/MD-2/CD14 HEK293 human Toll-like receptor 4NBP2- DMEM (4.5 g/L glucose) + 10% Reporter Cell Line (human (TLR4),human MD-2, human 26503/Novus FBS + 4 mM L-glutamine + 1 mM (HEK-TLR4)embryonic CD14, secreted alkaline Biologicals, LLC sodium pyruvate + 100unit/mL kidney- phosphatase (SEAP) reporter gene penicillin* + 100 μg/mLderived) under the transcriptional control streptomycin* + 10 μg/mL of aNF-κB response element blastcidin*^(†) + 2 μg/mL puromycin*† + 200 μg/mLzeocin*^(†) + 500 μg/mL G418*^(†) TLR2 Reporter Cell HEK293 humanToll-like receptor 2 NBP2- DMEM (4.5 g/L glucose) + 10% Line (HEK-TLR2)(human (TLR2), secreted alkaline 26274/Novus FBS + 4 mM L-glutamine + 1mM embryonic phosphatase (SEAP) reporter gene Biologicals, LLC sodiumpyruvate + 100 unit/mL kidney- under the transcriptional controlpenicillin* + 100 μg/mL derived) of a NF-κB response elementstreptomycin* + 10 μg/mL blastcidin*^(†) + 500 μg/mL G418*^(†) *Added,if necessary ^(†)Selection reagent11-3 Testing method11-3-1 Study on antagonistic (antagonist-like) action of olanexidine onTLR-4

-   -   Cell used: HEK293 cells expressing TLR4    -   Medium used

Preculture: DMEM+FBS (final concentration: 10%)+penicillin (finalconcentration: 100 units/mL) streptomycin (final concentration: 100μg/mL)

Sample administration and culture after administration: DMEM+FBS (finalconcentration: 5%)

-   -   Activity measurement: SEAP assay kit (manufactured by Novus        Biologicals)    -   Protein quantification: BCA protein assay (manufactured by        Funakoshi Co., Ltd.)        [1] Cells were adjusted to 1.0×10⁵ cells/well/100 μL with DMEM        containing 10% FBS, seeded to a 96-well plate (collagen-coated),        and cultured for 40 hours (37° C., 5% CO₂).        [2] 1.5% OPB was diluted to the concentrations shown in Table 8        below using 5% FBS.

TABLE 8 OPB concentration (μg/mL) Preparation concentration 20, 10, 5, 2Final concentration 10, 5, 2.5, 1[3] LPS was prepared at 20 ng/mL (final concentration: 10 ng/mL) using5% FBS.[4] After removal of the medium, media were added to the cells in theorder of 50 μL of the OPB medium and 50 μL of the LPS medium, followedby culture for 8 hours (37° C., 5% CO₂).[5] After the culture, 50 μL of the supernatant was transferred to eachwell of another 96-well plate, and SEAP assay was conducted.[6] 50 μL of 0.1% SDS-0.1 N NaOH was added to each well of the 96-wellplate from which the remaining supernatant was removed, and frozenovernight (−20° C.). After thawing, BCA protein assay was conducted.[7] The expression level of the SEAP reporter gene was calibrated withthe protein concentration to calculate an inhibition rate at each OPBconcentration.11-3-2 Study on antagonistic (antagonist-like) action of olanexidine onTLR-2

-   -   Cell used: HEK293 cells expressing TLR2    -   Medium used

Preculture: DMEM+FBS (final concentration: 10%)+penicillin (finalconcentration: 100 units/mL) streptomycin (final concentration: 100μg/mL)

Sample administration and culture after administration: DMEM+FBS (finalconcentration: 1%)

-   -   Activity measurement: SEAP assay kit (manufactured by Novus        Biologicals)    -   Protein quantification: BCA protein assay (manufactured by        Funakoshi Co., Ltd.)        [1] Cells were adjusted to 1.0×10⁵ cells/well/100 μL with DMEM        containing 10% FBS, seeded to a 96-well plate (collagen-coated),        and cultured for 36 hours (37° C., 5% CO₂).        [2] 1.5% OPB was diluted to the concentrations shown in Table 8        above using 1% FBS.        [3] LTA was prepared at 2 μg/mL (final concentration: 1 μg/mL)        using 1% FBS.        [4] After removal of the medium, media were added to the cells        in the order of 50 μL of the OPB medium and 50 μL of the LTA        medium, followed by culture for 12 hours (37° C., 5% CO₂).        [5] After the culture, 50 μL of the supernatant was transferred        to each well of another 96-well plate, and SEAP assay was        conducted.        [6] 50 μL of 0.1% SDS-0.1 N NaOH was added to each well of the        96-well plate from which the remaining supernatant was removed,        and frozen overnight (−20° C.). After thawing, BCA protein assay        was conducted.        [7] The expression level of the SEAP reporter gene was        calibrated with the protein concentration to calculate an        inhibition rate at each OPB concentration.        11-4 Results

The results are shown in FIG. 20.

The inhibition rate of SEAP was enhanced with elevation in OPBconcentration (IC₅₀: approximately 10 μg/mL), demonstrating that OPBexhibits antagonistic (antagonist-like) action on TLR4 and TLR2. Theseresults suggested that OPB has anti-inflammatory action by inhibitingimmune response mediated by TLR4 and TLR2.

Example 12

12. Study on anti-inflammatory action of olanexidine using mousemacrophage-like cell line RAW264.7

A mouse macrophage-like cell line RAW264.7, when irritated with LPS orLTA, starts immune response via a receptor recognizing it, to produce aninflammatory mediator NO. Accordingly, whether olanexidine would haveanti-inflammatory action on an inflammation due to LPS irritation wasconfirmed by using NO production from RAW264.7 cells as an indicator.

12-1 Test substance

Designation: 1.5% OPB

Formula: olanexidine gluconate . . . 1.5 w/v %

12-2 Cell

The cells described in Table 9 below were used.

TABLE 9 Designation Animal species Tissue Catalog No./supplier MediumRAW 264.7 Mouse, BALB/c Leukemic monocyte EC91062702-F0/DS Pharma DMEM +10% FBS + 100 Biomedical Co., Ltd. unit/mL penicillin* + 100 μg/mLstreptomycin* *Added, if necessary12-3 Testing method12-3-1 Study on anti-inflammatory action of olanexidine on LPSirritation

-   -   Cell used: RAW264.7    -   Medium used

Preculture: DMEM+FBS (final concentration: 10%)+penicillin (finalconcentration: 100 units/mL) streptomycin (final concentration: 100μg/mL)

Sample administration and culture after administration: DMEM+FBS (finalconcentration: 5%)

-   -   Activity measurement: Nitrate/Nitrite Colorimetric Assay Kit        (manufactured by Griess Reagents)    -   Protein quantification: not measured because the cells were        difficult to stain and destabilized values.        [1] Cells were adjusted to 1.0×10⁵ cells/well/100 μL with DMEM        containing 10% FBS, seeded to a 96-well plate (uncoated), and        cultured for 24 hours (37° C., 5% CO₂).        [2] 1.5% OPB was diluted to the concentrations shown in Table 8        above using 5% FBS.        [3] LPS was prepared at 200 ng/mL (final concentration: 100        ng/mL) using 5% FBS.        [4] After removal of the medium, media were added to the cells        in the order of 50 μL of the OPB medium and 50 μL of the LPS        medium, followed by culture for 8 hours (37° C., 5% CO₂).        [5] After the culture, 50 μL of the supernatant was transferred        to each well of another 96-well plate, and Nitrate/Nitrite        colorimetric assay was conducted.        12-3-2 Study on Anti-Inflammatory Action of Olanexidine on E.        coli (LPS-Producing Bacterium) Irritation    -   Cell used: RAW264.7    -   Medium used

Preculture: DMEM+FBS (final concentration: 10%)+penicillin (finalconcentration: 100 units/mL) streptomycin (final concentration: 100μg/mL)

Sample administration and culture after administration: DMEM+FBS (finalconcentration: 1%)

-   -   Activity measurement: Nitrate/Nitrite Colorimetric Assay Kit        (manufactured by Griess Reagents)    -   Protein quantification: not measured because the cells were        difficult to stain and destabilized values.        [1] Cells were adjusted to 1.0×10⁵ cells/well/100 μL with DMEM        containing 10% FBS, seeded to a 96-well plate (uncoated), and        cultured for 24 hours (37° C., 5% CO₂).        [2] 1.5% OPB was prepared at the concentrations shown in Table 8        above using 1% FBS.        [3] After removal of the medium, 50 μL of the OPB medium was        added to the cells, which were then left standing at 37° C.        under 5% CO₂.        [4] E. coli cultured overnight in MHB was prepared at McF 1 and        diluted 300-fold with 5% DMEM. Further, ampicillin was added        thereto so as to attain a final concentration of 50 μg/mL.        [5] The prepared bacterial culture medium was further added at        50 μL/well to the plate supplemented with the OPB medium, and        cultured for 24 hours (37° C., 5% CO₂) with the plate        hermetically sealed.        [6] After the culture, 50 μL of the supernatant was transferred        to each well of another 96-well plate, and Nitrate/Nitrite        colorimetric assay was conducted.        12-4 Results

The results are shown in FIG. 21.

NO production was decreased with elevation in OPB concentration,demonstrating that OPB exhibits NO production-inhibiting action (IC₅₀:approximately 10 μg/mL). These results suggested that OPB hasanti-inflammatory action.

INDUSTRIAL APPLICABILITY

The present invention provides a composition for amelioration and/orprevention of an inflammation, which is applicable to a wide range ofinflammatory diseases. Moreover, use of the composition of the presentinvention as a composition for amelioration and/or prevention of oralmucositis due to treatment of a cancer can prevent reduction in QOL,such as inhibition of a communication function, sleep disorder, pain, ordysphagia (decreased dietary intakes), in a patient who is receivingchemotherapy and/or radiotherapy, or disturbance of dose conformity ofchemotherapy and/or radiotherapy. Therefore, the present invention hashigh industrial usefulness.

The invention claimed is:
 1. A method for treating and/or preventing aninflammation, comprising administering a therapeutically effectiveamount of a composition for treatment and/or prevention of aninflammation, comprising olanexidine or a pharmacologically acceptablesalt thereof to a patient in need of treatment and/or prevention of aninflammation, wherein the inflammation is selected from stomatitis,gingivitis, and pneumonia.
 2. The method according to claim 1, whereinthe olanexidine or the pharmacologically acceptable salt thereof isolanexidine gluconate.
 3. The method according to claim 1, wherein thecomposition further comprises a poloxamer which is a block copolymerconsisting of a chain of polyoxypropylene (POP) and two chains ofpolyoxyethylene (POE) flanking the POP.
 4. The method according to claim3, wherein the poloxamer is selected from polyoxyethylene (42)polyoxypropylene (67) glycol, polyoxyethylene (54) polyoxypropylene (39)glycol, and polyoxyethylene (196) polyoxypropylene (67) glycol.
 5. Themethod according to claim 4, wherein the poloxamer is polyoxyethylene(42) polyoxypropylene (67) glycol.
 6. The method according to claim 1,wherein a concentration of the olanexidine gluconate in the compositionis 0.05 to 0.5% (W/V).
 7. The method according to claim 3, wherein aconcentration of the poloxamer in the composition is 0.1 to 5.0% (W/V).8. The method according to claim 1, wherein the composition is in a formof a liquid or a gargle.
 9. The method according to claim 2, wherein thecomposition further comprises a poloxamer which is a block copolymerconsisting of a chain of polyoxypropylene (POP) and two chains ofpolyoxyethylene (POE) flanking the POP.